The use of fluorescence technology has become widespread in the areas of clinical chemistry, i.e., laboratory testing and the medical diagnostic areas. The technology is particularly effective for making very sensitive and specific test determinations, competing effectively in many areas with radioimmunoassays and enzymatic immunoassays.
The phenomenon of fluorescence occurs when a molecule or atom is bombarded with light of given wavelengths; namely, the conversion of that light to an emission of light of a different wavelength. In macroscopic terms, the conversion is instantaneous, but in real terms the finite time differences between the absorption of the light by the molecule and the time interval during which the emitted light is given off is a measure of the characteristics of the bodies being measured.
The process of fluorescence starts with the absorption of light photons by atoms or molecules. The frequency of light absorption varies with the atom or molecule involved.
Fluorescent molecules in any specific environment have two characteristic spectra. The first, the so-called excitation spectrum, is represented by a series of wavelengths of light which are absorbed by the molecule with differing efficiencies. That is, out of a possible number of existing wavelengths which may be absorbed by the molecule to cause fluorescence, usually one of these will be absorbed at a greater level. Most atoms or molecules that absorb light convert this light energy into heat, but a few emit light or "fluoresce" at a lower light frequency. Photon absorption occurs rapidly in about 10.sup.-15 seconds. If the light excitation is abruptly interrupted, as with a very short pulse of light, photon light emission in the second spectrum will decay rapidly with a time constant that depends on the atom or molecule involved. The range of decay times is usually between 10.sup.-10 to 10.sup.-6 seconds (0.1 to 1000 nanoseconds). The intensity of the emission spectrum is directly proportional to the intensity of the exciting light.
It happens also that the intensity of the emitted light is also directly proportional to the concentration of the fluorescent molecules in the sample. It thus can be seen that a very sensitive technique for measuring the concentration of a fluorescent body can be evolved by controlling the intensity of the exciting light and other physical constants of the measuring system.
The analytical value of fluorescence decay time measurement arises from the fact that each atom or molecule has its own distinctive rate of decay. Each atom or molecule is excited at a different frequency and emits light only at a particular emission wavelength.
Analytical probes having fluorescent labels are valuable reagents for the analysis and separation of molecules and cells. Specific applications include: (1) identification and separation of subpopulations of cells in a mixture of cells by the techniques of fluorescence flow cytometry, fluorescence-activated cell sorting, and fluorescence microscopy; (2) determination of the concentration of a substance that binds to a second species (e.g., antigen-antibody reactions) in the technique of fluorescence immunoassay; (3) localization of substances in gels and other insoluble supports by the techniques of fluorescence staining. These techniques are described by Herzenberg et al., "Cellular Immunology," 3rd ed., chapt. 22, Blackwell Scientific Publications, 1978 (fluorescence-activated cell sorting); and by Goldman, "Fluorescence Antibody Methods," Academic Press, New York, 1968 (fluorescence microscopy and fluorescence staining).
When using fluorescers for the above purposes, there are many constraints on the choice of the fluorescer. One constraint is the absorption and emission characteristics of the fluorescer. Many ligands, receptors, and materials associated with such compounds in the sample in which the compounds are found, e.g., blood, urine, and cerebrospinal fluid, will fluoresce and interfere with an accurate determination of the fluorescence of the fluorescent label. Another consideration is the ability to conjugate the fluorescer to ligands and receptors and the effect of such conjugation on the fluorescer. In many situations, conjugation to another molecule may result in a substantial change in the fluorescent characteristics of the fluorescer and in some cases, substantially destroy or reduce the quantum efficiency of the fluorescer. A third consideration is the quantum efficiency of the fluorescer. Also of concern is whether the fluorescent molecules will interact with each other when in close proximity, resulting in self-quenching. An additional concern is whether there is non-specific binding of the fluorescer to other compounds or container walls, either by themselves or in conjunction with the compound to which the fluorescer is conjugated.
The value of the methods indicated above are closely tied to the availability of suitable fluorescent compounds. In recent years, the evolution of solid state emitting diodes and solid state detectors has progressed rapidly as has the chemistry of fluorescent dyes. This evolution opened several opportunities for applications in the red and near infrared region (617 to 2500 nm). The red and near infrared region appears particularly suitable for biological analysis because of the low background fluorescence generated by biological material or by chemical compounds. Moreover, the development of inexpensive commercial diode lasers with emitting wavelengths of 670, 750, 780, and 810 nm, led to research into dyes that can be excited at these wavelengths.
Earlier efforts in this field produced many dyes, most of them belonging to the cyanine family. However, these dyes did not satisfy all the requirements needed for useful applications. Waggoner et al. (Bioconjugate Chemistry, 4, 105-111 (1993)) studied the chemistry of the cyanine dyes in order to develop conjugation sites and also to enhance water solubility. They synthesized sulfoindocyanine dyes with high hydrosolubility and reactive groups available for conjugation with biological compounds. However, these dyes cannot be excited by diode lasers and/or have low fluorescence quantum yields.
During the development of photo-sensitive dyes, a Kodak research group added one more ring to the indolenine moiety and made the dye structure more rigid by means of an additional ring in the polymethine chain. Trying to reach the same goal, Patonay et al. (J. Org. Chem., 57, 4578-4580 (1992)) studied structures bearing activated groups for conjugation purposes and a chlorocyclohexenyl ring in the polymethine chain. The latter increases the rigidity of the structure, thus enhancing the fluorescence quantum yield of the dye. It also provides a convenient site for chemical substitution at the central ring, useful for introducing reactive groups or electron withdrawing radicals capable of modifying the excitation wavelength. Some of the drawbacks of Kodak's and Patonay's dyes are their low hydrophilicity and/or the mismatch with commercially available diode lasers.
The prior art is silent regarding the sulfo benz[e]indocyanine fluorescent dyes of the present invention as well as their uses in immunoassays, DNA probes, DNA sequencing, HPLC, capillary electrophoresis, fluorescence polarization, total internal reflection fluorescence, flow cytometry, and optical sensors. The above analytical techniques would enjoy substantial benefits when using the fluorescent dyes of the present invention, which have high quantum efficiency, absorption, and emission characteristics at the red and near infrared region, simple means for conjugation, and are substantially free of non-specific interference.